The present invention relates to a method of purifying interferon alpha. More particularly, the present invention relates to a method employing hydrophobic electrolyte solutions throughout the purification scheme so as to preserve native isospecies composition of interferon alpha.
Interferons are proteins with distinctive biological properties, the most remarkable of which is the capability of rendering living cells resistant to viral infection. Interferons are produced by cells in response to certain inducers such as a virus, a mitogen or a double stranded RNA. Currently, three major types of human interferons are known, each differentiated according to the initial producer cell and the inducer applied. They are interferons alpha (leukocyte), beta (fibroblast) and gamma (immune). Within the alpha and beta types, there are multiple subspecies coded by separate interferon genes. Interferon gamma, however, is a product of a single gene; but as a result of a postranslational modification, it exists in various forms.
Human interferon alpha is the most complex family of interferons. The heterogeneity of isospecies of highly purified preparations is well established and is revealed by many chromatographic media, as well as SDS-PAGE or isoelectric focusing. The amino acid composition of different interferon alpha isospecies shows significant similarities, and some of them can be considered identical. However, biological activity of the isospecies is quite different and varies with the cells used for assay. It seems that different isospecies are addressed to different types of cells. In other words, the bioresponse to interferons may be critically dependent upon the presence of different isospecies in the appropriate ratio.
In order to obtain full therapeutic effect of interferon alpha, it is important to preserve all isospecies during the purification procedure.
There are many methods for interferon alpha purification, using combinations of classical chromatography techniques, monoclonal antibodies, high performance liquid chromatography, etc., in a unique sequence of steps. By using different techniques, different degrees of purity and different compositions of isospecies are achieved.
The classical purification method of interferon alpha described by Cantell (Methods in Enzymology 78:499-505, 1981) employs relatively harsh conditions. This method uses differential acid-ethanol precipitation within a narrow pH range, a difficult step to control, largely because of nonaqueous conditions (high concentration of ethanol) and low temperature requirements. A large variation of interferon isospecies can result from batch-to-batch. Another purification method is described by Chadha et al (U.S. Pat. No. 4,485,038) which preferentially recovers the pH labile form of interferon alpha. A drawback, however, is that the mild conditions of the purification allow possible viral contaminations, and in addition some isospecies are eliminated from the final preparation.
There are several more purification procedures which apply harsh conditions for the recovery of interferon causing inactivation of some of interferon alpha isospecies.
As the pool of leukocytes for production of interferons increases, so also does the possible contamination with exogenous microorganisms. Contamination of the leukocytes with viruses which may derive from apparently healthy individuals who donate blood is of great concern. Infections with hepatitis A, B or non-A and non-B are good examples. However, cells can also be contaminated by other DNA and RNA viruses known to infect man. These serious contaminants include, among others, the viruses responsible for chronic infections such as cytomegalo and Epstein Barr viruses, members of the herpes virus family.
More recently, retroviruses of the Lentivirinae subfamily (Human Immuno-deficiency Virus [HIV] [International Committee on the Toxonomy of Viruses. Science 232:697, 1981], previously known as LAV-I/HTLV-III) present a serious problem source of contamination. Although the U.S. Food and Drug Administration regulations require that leukocytes must come from blood which is negative for hepatitis B surface antigen and HTLV-III antibody, these precautions may not be sufficient. There are cases where viremia occurs without the presence of antibodies.
Furthermore, Montagnier et al (Science 232:343, 1986) recently have found a new AIDS virus with an antigenic structure so different from the prototype that "classical" antigens did not react with antibodies to the new virus, and antibodies against the "classical" strain of the HIV viruses did not recognize the antigens of the new isolate. In other words, some of the viruses from the retrovirus family may escape detection at screening. Thus, they may contaminate batches of starting leukocytes.
Since viremia cannot be assessed routinely by blood banks, there will always be a risk factor when the natural product derived from donated blood is used, unless the purification contains steps which are known to eliminate viruses.
The most effective way to eliminate viral contamination from interferon batches is the destruction of the virus and its nucleic acid. In the Cantell preparation, the goal is obtained by low pH treatment in the presence of ethyl alcohol. Unfortunately, such treatment has a negative impact on the composition of interferon isospecies in the final preparation.
Concerning the above, the present invention focuses on:
(a) elimination of exogenous Sendai virus (the interferon alpha inducer), as well as other endogenous viruses; and PA1 (b) preservation of a natural composition of interferon alpha isospecies in the final preparation.